Browsing by Subject "laser capture microdissection"

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    Gene Expression Analysis in Neurons throughout Late-Onset Alzheimer’s Disease Pathological Progression
    (2017-05-04) Bonawitz, Kirsten
    Over 20 susceptibility loci for late-onset Alzheimer’s disease (LOAD) have been identified in large-scale genome-wide association studies (GWAS), and several past studies have found differences in gene expression between normal and AD brain tissue. Several limitations exist in this previous research, including the use of whole brain tissue and comparing control brain tissue to AD brain tissue, which provides minimal knowledge about which genes play a critical role in the early stages of the disease. To overcome these limitations, the aims of this study were: (1) to develop and optimize a method to isolate single cells from frozen brain tissue while preserving RNA for downstream gene expression analysis and (2) to utilize this method to analyze gene expression in neurons over the course of LOAD pathological progression. We combined immunohistochemistry with laser capture microdissection (LCM) to collect single neurons from normal, mild-cognitive impairment (MCI), mild AD, and severe AD frozen human temporal cortex tissues. Gene expression was determined using the NanoString nCounter Single Cell gene expression assay. Analyzed samples showed at least 10-fold neuronal enrichment, validating the collection of homogenous pools of neurons. Our results confirm that for many LOAD-associated genes, mRNA levels indeed vary throughout disease progression. Notably, APOE mRNA levels were found to increase throughout LOAD pathological progression, while APP mRNA levels were elevated in the AD samples but not in the MCI samples. These results suggest that regulation of APOE may contribute to the development of LOAD while APP regulation may become altered only once the disease has progressed. This endeavor diverges from past studies by examining a single cell type (neurons) as opposed to whole brain tissue, allowing us to gain accuracy and specificity in identifying target genes. Moreover, findings in MCI tissue implicate the expression regulation of critical genes in playing a part in the early stages of disease, suggesting a role in causing LOAD.